FOLDING KINETICS OF THE SH3 DOMAIN OF PI3 KINASE BY REAL-TIME NMR COMBINED WITH OPTICAL SPECTROSCOPY

The refolding kinetics of the chemically denatured SH3 domain of phosp hatidylinositol 3'-kinase (PI3-SH3) have been monitored by real-time o ne-dimensional H-1 NMR coupled with a variety of other biophysical tec hniques. These experiments indicate that the refolding kinetics of PI3 -SH3 are biphasic. The slow phase (27 (+/-8)% amplitude) is due to a p opulation of substantially unfolded molecules with an incorrectly conf igured cis proline residue. The fast phase (73 (+/-8)% amplitude) corr esponds to the folding of protein molecules with proline residues in a tuans configuration in the unfolded state. NMR experiments indicate t hat the first species populated after the initiation of folding exhibi t poor chemical shift dispersion and have spectra very similar to that of the denatured protein in 8 M guanidine hydrochloride. Linear combi nations of the first spectrum and of the spectrum of the native protei n accurately reconstruct all of the spectra acquired during refolding. Consistent with this, native side-chain and backbone H-alpha atom pac king (NMR), secondary structure (far-UV circular dichroism), burial of aromatic residues (near-UV circular dichroism), intrinsic fluorescenc e and peptide binding activity are all recovered with effectively iden tical kinetics. Equilibrium unfolding and folding/unfolding kinetics y ield,within experimental error, identical values for the free energy o f unfolding (Delta G(u-H2O) = 3.38 kcal mol(-1)) and for the slope of the free energy of unfolding versus denaturant concentration (m(eq) = 2.33 kcal mol(-1) M-1). Together, these data provide strong evidence t hat PI3-SH3 folds without significant population of kinetic well-struc tured intermediates. That PI3-SH3 folds slowly (time constant 2.8 seco nds in H2O at 20 degrees C) indicates that slow refolding is not alway s a consequence of kinetic traps but may be observed even when a prote in appears to fold via a simple, two-state mechanism. (C) 1998 Academi c Press Limited.